Abstract Thymocyte development is tightly regulated, requiring successful transit of cells through several developmental stages and checkpoints prior to thymic egress. Each checkpoint of thymocyte development, involves induction or repression of a particular set of genes. Disruptions in gene regulation leads to developmental arrest, a failure to generate T cells and deficits in adaptive immunity. Gene expression is coordinated by transcriptional activators, repressors, and chromatin modifiers. In general, histone acetylation promotes gene expression while histone deacetylation leads to repression. We investigated the role of histone deacetylase-3 (HDAC3) in T cell development using CD2-icre conditional knockout (HDAC3- cKO) mice. Although T cells co-express several HDAC family members during development, these other HDAC family members cannot compensate for the loss of HDAC3 as HDAC3-cKO mice have a block in T cell development at the DP stage due to an inability to undergo positive selection. The block in T cell development could not be rescued by an OT-II TCR transgene, or by a Bcl-2 or Bcl-xl transgene. Successful positive selection requires down-regulation of ROR?t, as mice with constitutive expression of ROR?t have a similar block in T cell development at positive selection. In HDAC3-cKO mice, ROR?t was not down-regulated upon TCR stimulation at the DP stage, demonstrating that the block in positive selection may be due to an inability to down-regulate ROR?t. Consistent with this, we observed enhanced RORC promoter acetylation in the absence of HDAC3, indicating that HDAC3 may directly deacetylate histones at the RORC promoter to inhibit expression. ROR?t also controls expression of Bcl-xl at the DP stage to regulate DP survival. Therefore, to determine whether sustained expression of ROR?t was responsible for the block in positive selection, ROR?t-KO Bcl-xl Tg HDAC3-cKO (hereafter called RB3) mice were generated, which restored positive selection leading to the generation of normal numbers of CD8SP thymocyte (TCR?+, Runx3+) numbers. However, CD4SP thymocytes were not generated, indicating a potential role for HDAC3 in lineage choice during T cell development, which we believe is due to a defect in CD4 expression during positive selection and lineage commitment. To confirm the altered lineage commitment, we generated OT-II RB3 mice and found that the OT-II TCR-expressing thymocytes are skewed towards the CD8 lineage rather than CD4SP T cells as is observed in OT-II transgenic (otherwise WT) mice. The focus of this proposal is to define the role of HDAC3 in positive selection and CD4 lineage development